Background: early blight disease caused by Alternaria solani is one of the most destructive diseases of the tomato, reducing tomato production globally. Methods: four fungal isolates were collected from four tomato cultivars and identified through morphological characterization and polymerase chain reaction (PCR) amplification of the internal transcript spacer (ITS) region. Plectranthus neochilus and Parthenocissus quinquefolia methanol extracts and the bioagents Trichoderma viride and Pseudomonas fluorescens were used as antifungal agents in vitro and in vivo and compared with chlorothalonil, a reference chemical fungicide. HPLC analysis of the plant extracts was used to identify the main flavonoid compounds, namely, rutin and myricetin. Results: molecular characterization showed that the fungal isolates belonged to A. solani. The results of in vitro antifungal activity studies revealed that chlorothalonil, at a concentration of 2500 mg/L, showed the highest inhibition percentage of fungal growth (IPFG) against A. solani (84.4%), followed by the bioagents T. viride and P. fluorescens, with IPFG values of 72.9% and 67.9%, respectively. Moderate to weak activity was found against A. solani when P. neochilus and P. quinquefolia extracts were applied at a concentration of 2500 mg/L, with an IPFG value of 54% for both extracts. The results of in vivo spray application showed that T. viride and chlorothalonil, as well as P. fluorescens, significantly reduced the disease index of early blight, and followed by the P. neochilus and P. quinquefolia extracts. By HPLC, the flavonoid compounds rutin and myricetin were identified in P. neochilus (leaf) with amounts of 2429.60 and 75.92 mg/100 g of extract, and in P. quinquefolia (fruit), with amounts of 1891.60 and 241.06 mg/100 g of extract, respectively. Conclusions: the results of the bioactivity of plant extracts and the bioagents indicate a vital role as antifungal activity against A. solani.